In studying the relationship between structure and function of muscle, we have established that the structure of the myosin filaments is a reflection of the atomic structure of the myosin head. The open/closed switch 2 conformations in the myosin head is closely correlated with the disordered/ordered helical array of heads on the surface of the myosin filament. Only the myosin heads in the ordered array (in the switch 2 closed state) are poised to generate force. To our knowledge, this is the first time that the atomic structures and the biochemical states of myosin and the structure of the myosin filaments are shown to be tightly coupled. [unreadable] We are pursuing other factors that could affect the structure of the myosin filaments that are relevant to force generation. Phosphorylation of the myosin regulatory light chain (RLC) in the skeletal and cardiac myosin is correlated with enhancement of tension at submaximal level of activation and reduces the effect of sarcomere length on force. It has been suggested that phosphorylation disorders the myosin filament, moving myosin heads away from the myosin filament, such that the closer proximity improves the probability of interaction with actin. Phosphorylation of the RLC could, therefore, lead to a structural pathway in modulating force levels independent of ATP hydrolysis. Structural studies have been limited to isolated filaments. The key factors for force generation such as the number of cross-bridges attached, the filament structures and the lattice spacing have to be investigated using muscle cells. During FY2008, X-ray diffraction from permeabilized muscle was obtained to determine the degree of orderliness of the filaments. The results showed that phosphorylation of the RLC brought about disorder. The degree of disorder was directly correlated with the level of phosphorylation. It also changes the separation distances between the filaments and the fraction of myosin heads bound to actin in a relaxed muscle. The latter result could provide an explanation of increased calcium sensitivity by RLC phosphorylation, particularly in cardiac muscle. Several control experiments are in the planning to be carried out. [unreadable] During FY2008, preliminary study was conducted on the mechanical and biochemical properties of fast muscle fibers from a mouse model of a genetic disease, the Pompe disease. Also known as type II glycogen storage disease, it is caused by lysosomal alpha-glucosidase deficiency which leads to cardiomyopathy and skeletal muscle myopathy. Protein distributions and force levels from the GAA knockout and the wild type have been compared. Further experiments are needed to confirm the results.